The principal objective of this proposal is to use T4 lysozyme as a model system to better understand the factors that determine the folding, stability, structure and function of proteins. The specific research to be accomplished includes the following: (a) An attempt will be made to simplify the protein folding problem by identifying which residues, or combinations of residues, in T4 lysozyme are critical for folding and stability. We want to understand not only how given residues contribute to stability, but also the signals, if any, that define the elements of secondary structure. Ultimately we would like to reduce the amino acid sequence of T4 lysozyme to the simplest form that will still give a folded, functional protein. (b) Methionine substitution, together with other nonpolar replacements, will be used to better understand the core-packing interactions that are critical to protein folding. (c) Methods will be developed and tested to improve protein stability. (d) Cavities within T4 lysozyme will be exploited both to understand protein-ligand interaction and to engineer novel active sites. (e) The role of strain within the protein will be systematically analyzed.